HNF4 and HNF1 as well as a panel of hepatic functions are extinguished and reexpressed in parallel in chromosomally reduced rat hepatoma-human fibroblast hybrids.

Rat hepatoma-human fibroblast hybrids of two independent lineages containing only 8-11 human chromosomes show pleiotropic extinction of thirteen out of fifteen hepatic functions examined. Reexpression of the entire group of functions most often occurs in a block, and except for one discordant subclone, correlates with loss of human chromosome 2. The extinguished cells and their reexpressing derivatives have been examined for the expression of seven liver-enriched transcription factors. C/EBP, LAP, DBP, HNF3, and vHNF1 expression are not systematically extinguished in parallel with the hepatic functions. However, HNF1 and HNF4 show a perfect correlation with phenotype: these factors are expressed only in the cells showing pleiotropic reexpression. Since recent evidence indicates that HNF4 controls HNF1 expression, it can be proposed that the HNF4 gene is the primary target of the pleiotropic extinguisher.

Dynamic molecular combing: stretching the whole human genome for high-resolution studies.

DNA in amounts representative of hundreds of eukaryotic genomes was extended on silanized surfaces by dynamic molecular combing. The precise measurement of hybridized DNA probes was achieved directly without requiring normalization. This approach was validated with the high-resolution mapping of cosmid contigs on a yeast artificial chromosome (YAC) within yeast genomic DNA. It was extended to human genomic DNA for precise measurements ranging from 7 to 150 kilobases, of gaps within a contig, and of microdeletions in the tuberous sclerosis 2 gene on patients' DNA. The simplicity, reproducibility, and precision of this approach makes it a powerful tool for a variety of genomic studies.

Identification of the tuberous sclerosis gene TSC1 on chromosome 9q34.

Tuberous sclerosis complex (TSC) is an autosomal dominant disorder characterized by the widespread development of distinctive tumors termed hamartomas. TSC-determining loci have been mapped to chromosomes 9q34 (TSC1) and 16p13 (TSC2). The TSC1 gene was identified from a 900-kilobase region containing at least 30 genes. The 8.6-kilobase TSC1 transcript is widely expressed and encodes a protein of 130 kilodaltons (hamartin) that has homology to a putative yeast protein of unknown function. Thirty-two distinct mutations were identified in TSC1, 30 of which were truncating, and a single mutation (2105delAAAG) was seen in six apparently unrelated patients. In one of these six, a somatic mutation in the wild-type allele was found in a TSC-associated renal carcinoma, which suggests that hamartin acts as a tumor suppressor.

Recommendations for locus-specific databases and their curation.

Expert curation and complete collection of mutations in genes that affect human health is essential for proper genetic healthcare and research. Expert curation is given by the curators of gene-specific mutation databases or locus-specific databases (LSDBs). While there are over 700 such databases, they vary in their content, completeness, time available for curation, and the expertise of the curator. Curation and LSDBs have been discussed, written about, and protocols have been provided for over 10 years, but there have been no formal recommendations for the ideal form of these entities. This work initiates a discussion on this topic to assist future efforts in human genetics. Further discussion is welcome.

Estimating the birth prevalence and pregnancy outcomes of congenital malformations worldwide.

Congenital anomaly registries have two main surveillance aims: firstly to define baseline epidemiology of important congenital anomalies to facilitate programme, policy and resource planning, and secondly to identify clusters of cases and any other epidemiological changes that could give early warning of environmental or infectious hazards. However, setting up a sustainable registry and surveillance system is resource-intensive requiring national infrastructure for recording all cases and diagnostic facilities to identify those malformations that that are not externally visible. Consequently, not all countries have yet established robust surveillance systems. For these countries, methods are needed to generate estimates of prevalence of these disorders which can act as a starting point for assessing disease burden and service implications. Here, we describe how registry data from high-income settings can be used for generating reference rates that can be used as provisional estimates for countries with little or no observational data on non-syndromic congenital malformations.

Molecular cloning of the human Goodpasture antigen demonstrates it to be the alpha 3 chain of type IV collagen.

To characterize the autoantigen of Goodpasture's (anti-glomerular basement membrane) disease, a molecule of 26-kD reactive with autoantibodies from patients' sera was purified from collagenase digests of sheep glomerular basement membrane. Short internal amino acid sequences were obtained after tryptic or cyanogen bromide cleavage, and used to deduce redundant oligonucleotides for use in the polymerase chain reaction on cDNA derived from sheep renal cortex. Molecules of 175 bp were amplified and found to come from two cDNA sequences. One was identical to that of a type IV collagen chain (alpha 5) cloned from human placenta and shown to be expressed in human kidney. The other was from a type IV collagen chain with close similarities to alpha 1 and alpha 5 chains, and was used to obtain human cDNA sequences by cDNA library screening and by further polymerase chain reaction amplifications. The correspondence of the derived amino acid sequence of the new chain with published protein and cDNA sequences shows it to be the alpha 3 chain of type IV collagen. Its gene, COL4A3, maps to 2q36-2q37. The primary sequence and other characteristics of this chain confirm that it carries the Goodpasture antigen.

Familial distal renal tubular acidosis is associated with mutations in the red cell anion exchanger (Band 3, AE1) gene.

All affected patients in four families with autosomal dominant familial renal tubular acidosis (dRTA) were heterozygous for mutations in their red cell HCO3-/Cl- exchanger, band 3 (AE1, SLC4A1) genes, and these mutations were not found in any of the nine normal family members studied. The mutation Arg589--> His was present in two families, while Arg589--> Cys and Ser613--> Phe changes were found in the other families. Linkage studies confirmed the co-segregation of the disease with a genetic marker close to AE1. The affected individuals with the Arg589 mutations had reduced red cell sulfate transport and altered glycosylation of the red cell band 3 N-glycan chain. The red cells of individuals with the Ser613--> Phe mutation had markedly increased red cell sulfate transport but almost normal red cell iodide transport. The erythroid and kidney isoforms of the mutant band 3 proteins were expressed in Xenopus oocytes and all showed significant chloride transport activity. We conclude that dominantly inherited dRTA is associated with mutations in band 3; but both the disease and its autosomal dominant inheritance are not related simply to the anion transport activity of the mutant proteins.

Association of herpes simplex thymidine kinase gene with chromosome No. 18 in transformed human cells.

The herpes simplex virus type-2 (HSV-2)-transformed human cell line HB-2-3 was fused with thymidine kinase (TK)-deficient mouse cells [LM(TK-)], and 12 independent hybrids were isolated with the use of the HAT (hypoxanthine, amethopterin, and thymidine)-ouabain selection system. Discontinuous polyacrylamide gel electrophoresis studies demonstrated that the HSV-2-specific TK was the selected enzyme in the hybrids. Isoenzyme analysis and karyotyping were used in the analysis of the hybrids for the presence of human chromosomes. All 12 hybrids contained human chromosome No. 18 and the enzyme peptidase A, which is encoded by a gene on this chromosome. Hybrids were exposed to bromodeoxyuridine (BrdUrd) as a means of selection for cells that had lost HSV-2 TK activity. Isoenzyme and karyotyping data obtained from 33 BrdUrd-resistant sublines were consistent with the hypothesis that the HSV-2 TK gene is associated with chromosome No. 18 in the HB-2-3 cell line.

Human lymphoma-lymphoma hybrids and lymphoma-leukemia hybrids. I. Isolation, characterization, cell surface markers, and B-cell markers.

Four new somatic cell hybrids were obtained by fusion of various Burkitt's lymphoma (BL)-derived cell lines that had different selective markers: Raji-P3HR-1, Daudi-Raji, and a P3HR-1-P3HR-1 "autohybrid" derived from two P3HR-1 sublines. In addition, a hybrid was obtained between the Daudi (BL) line and the human leukemia cell line K562. The hybrids were extensively characterized by means of chromosome, isozyme, and HLA surface markers. The phenotypic differences between the parent cell lines allowed some conclusions with respect to the expression of latent Epstein-Barr virus (EBV) genomes, C3 and EBV receptors, and of immunoglobulin and beta 2-microglobulin-HLA expression as well as the influence of the leukemia cell (K562) genome on B-cell properties in the Daudi-K562 hybrid. B-cell and differentiated markers of these hybrids were characterized. High-level expression dominated for the marker C3 and EBV receptors, which showed a good correlation coefficient of 0.84, as was true for Fc receptors and surface immunoglobulin. The Daudi-K562 hybrid showed loss of all B-cell markers but retention of the leukemia cell markers (e.g., hemoglobin synthesis).

Hybridization of a myeloid leukemia-derived human cell line (K562) with a human Burkitt's lymphoma line (P3HR-1).

The myeloid leukemia-derived Epstein-Barr virus (EBV)-negative human lymphoid cell line K562 was successfully hybridized with the EBV-carrying Burkitt's lymphoma line P3HR-1. Authenticity of the hybrid PUTKO-1 was established by chromosome and isoenzyme studies. A virtually complete hybrid PUTKO-1 carried the EBV genome derived from the lymphoma parent. It averaged 26 EBV DNA copies per cell and was 100% positive for Epstein-Barr virus-associated nuclear antigen (EBNA). In most respects, the hybrid resembled the K562 parent: It had a high Fc receptor concentration, high sensitivity to natural killer cells, absence of EBV C3 receptors, and deficiency of membrane-associated beta 2-microglobulin (beta 2M) and HLA, in parallel with intracellular synthesis and secretion of beta 2M to the medium. Unlike the P3HR-1 parent, the hybrid was completely nonpermissive for antigens of the EBV cycle, early antigen, and viral capsid antigen. None of the 3 inducing agents, 5-lodo-2'-deoxyuridine, 12-O-tetradecanoyl-phorbol 13-acetate, or sodium butyrate, caused any viral antigen synthesis in PUTKO-1 in contrast to the good inducibility of the parental P3HR-1 subline. Thus the myeloid parent restricted expression of EBV antigens except EBNA. This exception further supports the concept that EBNA is an autonomous function of the viral genome, independent of host cell control that regulates expression of antigens related to the viral cycle. On the contrary, extinction of viral antigens in this hybrid between 2 cell lineages supports our previous concept that the ability to produce viral antigens is similar to a differentiated B-cell property.

Characteristics of four new human cell lines derived from squamous cell carcinomas of the head and neck.

Four human cell lines were established from biopsy specimens of squamous cell carcinomas of the larynx (TR131 and TR138), tongue (TR126), and buccal mucosa that had infiltrated a lymph node (TR146). All 4 lines readily formed colonies on a plastic substratum, but they were virtually incapable of forming colonies in an anchorage-independent semisolid support system of soft agar (cloning efficiencies, less than 0.02%). The proliferation of this group of tumor-derived cell lines, therefore, appeared to be highly anchorage dependent. Keratin filaments could be visualized in each line by indirect immunofluorescence with the use of polyclonal or monoclonal antibodies to keratins; staining with monospecific antibodies indicated that 3 of the 4 lines expressed simple epithelial keratins 8 and 18, whereas 1 of the 4 also expressed keratin 19. A panel of lectins revealed characteristic localization patterns distinct from those observed on other epithelial cell lines. Cells from 3 lines (TR131, TR138, and TR146) inoculated into nude mice (nu/nu) produced cystic nodules or unequivocal tumors having a histology indicating a squamous cell origin for the injected cells. Electron microscopy demonstrated that the cell lines covered a spectrum of differentiation capability ranging from the undifferentiated monolayer cultures of TR126 to the rather well differentiated, stratified cultures of TR131.

Tissue culture model of transitional cell carcinoma: characterization of twenty-two human urothelial cell lines.

Twenty-two continuous cell lines derived from normal and neoplastic urothelium, maintained under identical culture conditions, were characterized in terms of isozyme phenotype, tumorigenicity, and xenograft morphology following xenotransplantation to nude mice, cytological appearance, in vitro growth rate, labelling index, and colony-forming efficiency, in parallel with separate studies of in vitro drug sensitivities and monoclonal antibody reactivities. Three groups were identified: (a) distinct lines with differing isozyme patterns, a broad spectrum of growth characteristics, and xenograft morphologies similar to the histopathology of the parent tumors after periods of up to 17 yr following establishment in vitro; (b) cross-contaminated sublines (maintained separately in different laboratories for periods of up to 10 yr), with identical isozyme patterns and similar growth characteristics, but differing markedly in tumorigenicity and xenograft morphology; and (c) lines derived from normal urothelium which were nontumorigenic and had an isozyme pattern usually only encountered in untransformed cells. These data indicate that cell lines representative of human transitional cell carcinomas can be selected on the basis of xenograft morphology and isozyme patterns, and that a panel of lines derived from normal and neoplastic urothelium could provide a model system to study the biology and treatment of this disease.

Characteristics of a new human neuroblastoma cell line which differentiates in response to cyclic adenosine 3':5'-monophosphate.

A new human cell line, TR14 , has been established in tissue culture from biopsy material of a primary neuroblastoma tumor. Most TR14 cells have short processes and grow mainly in clumps adhering to cells attached to the substratum. TR14 cells form colonies in soft agar demonstrating anchorage independence of growth and produce tumors in nude mice with histologies similar to that of the patient's tumor. The neurotransmitter-synthesizing activity of these cells is predominantly cholinergic with only a minor adrenergic component, since the activity of choline acetyltransferase is about 20-fold greater than that of tyrosine hydroxylase. Treatment with N6,O2'-dibutyryl cyclic adenosine 3':5'-monophosphate induces TR14 neuroblastoma cells to extend fine, long processes or neurites. This morphological change is accompanied by elevated numbers of cytoplasmic dense-core vesicles observed by electron microscopy and an increase in the activities of neurotransmitter-synthesizing enzymes. Differentiation therefore occurs at the levels of cellular morphology, ultrastructure, and biochemistry. Prostaglandin E1 and cholera toxin can also induce differentiation, but a range of other agents including dimethyl sulfoxide, nerve growth factor, butyrate, corticosteroids, and 5-bromodeoxyuridine is ineffective. The concomitant induction of both morphological and biochemical differentiation therefore appears to be exclusively a cyclic adenosine 3':5'-monophosphate-mediated event in this cell line.

Integrated radiation hybrid and yeast artificial chromosome map of chromosome 9p.

A panel of 93 radiation-reduced hybrids have been screened using PCR amplification and oligonucleotide primers for sequence-tagged sites (STSs) specific for 114 single-copy loci mapping to the short arm of chromosome 9. An x-ray dose of 6,000 rads gave an average retention frequency of approximately 23%. We have constructed a framework map containing 31 markers ordered by analyzing coretention patterns, with support for the order greater than 1,000:1. In addition, we have placed the remaining markers which could not be mapped to a single interval with this support to a range of intervals on the framework map. The STS oligonucleotide primers used in the construction of the radiation hybrid (RH) map have been used to isolate and order yeast artificial chromosomes (YACs) assigned to 9p identified from the CEPH megaYAC library. Eighty-nine STS markers have screened positive with at least one YAC. A total of 88 individual YACs (with an average size of 0.9 MB) have been placed on the map in a series of contigs and in some cases mapped cytogenetically by fluorescence in situ hybridization. Additionally, the YAC information has been used in conjunction with the RH framework placements to generate an integrated map containing 65 loci including 51 uniquely positioned markers, with an average resolution of 0.79 Mb.

Isolation of a cDNA probe for a human jejunal brush-border hydrolase, sucrase-isomaltase, and assignment of the gene locus to chromosome 3.

We report the nucleotide sequence and derived amino acid sequence of a cDNA clone encoding most of the N-terminal, isomaltase region of human sucrase-isomaltase (SI). A plasmid containing this cDNA, pS12, identifies a 6-kb mRNA found in human jejunum and the human colon carcinoma cell line Caco-2. This human SI cDNA shows extensive overall homology with recently published rabbit SI cDNA. Using pS12 to probe DNA from a panel of somatic cell hybrids, we have assigned the gene encoding human SI to chromosome 3.

Development and characterization of cisplatin-resistant human testicular and bladder tumour cell lines.

Cisplatin-resistant cells were derived in vitro from a human bladder carcinoma cell line (RT112) and a testicular tumour cell line (SuSa) by continuous exposure to increasing concentrations of cisplatin for 14 and 11 months, respectively. Both resistant cell lines had a four-fold level of resistance relative to their parental cell lines, comparing the cisplatin concentration to inhibit colony forming ability by 70%. These levels of resistance were retained in the absence of cisplatin for at least 3 months. In each case, four-fold fewer micronuclei were produced in the resistant lines by the same cisplatin concentrations. Cross-resistance to carboplatin and methotrexate was observed in both resistant cell lines, but neither line was resistant to doxorubicin. Isozyme and DNA analysis with hypervariable probes confirmed the origin of each resistant cell line from its parental line. Population doubling times and intermitotic times were similar in each of the pairs of cell lines. Karyotyping showed that the resistant cell lines had gained and lost marker chromosomes, but there were no changes common to both resistant cell lines.

Analysis of the 5'-upstream regions of the human relaxin H1 and H2 genes and their chromosomal localization on chromosome 9p24.1 by radiation hybrid and breakpoint mapping.

Relaxins are known endocrine and autocrine/paracrine hormones that play a major role in reproduction. In the human there are two relaxin genes, H1 and H2 which share 90% sequence homology within their coding region. The biological and evolutionary significance of two highly homologous and biologically active human relaxins is unknown. In order to achieve a better understanding of the regulatory mechanisms involved in the differential expression of these two genes and to gain insight into their role(s) in the preterm premature rupture of the membranes, we have investigated the properties of their 5'-upstream regions and mapped them both by radiation hybrid and breakpoint mapping into the same chromosome 9p24.1 locus. The 5' ends of these relaxin genes could be divided into a proximal highly homologous segment and a distal non-homologous region. Within the proximal region are contained several putative regulatory elements common to both genes, suggesting a similar regulatory mechanism. The clustering of the relaxin genes within the same chromosomal locus suggests that these genes may be under a common regulation. On the other hand, a distinct gene-specific regulation may also exist for the individual relaxin genes since cis elements specific to each gene were identified at their 5' ends. Moreover, the observed divergence at the distal region of their 5'-upstream sequences may provide the structural features that act as gene-specific transcription regulators. Since the two genes are highly homologous in both their coding and flanking regions, the divergence at the distal region of their 5' ends may be important in the regulation of these genes and in their involvement in the pathology of preterm birth.

Molecular size of enzymes in Trypanosoma cruzi considered in relationship to the genetic interpretation of isozyme patterns.

The molecular size of seven Trypanosoma cruzi enzymes, chosen because of their frequent use in studying trypanosome populations, has been found to be similar to that of their mammalian equivalents. Malic enzyme (NADP-dependent malate dehydrogenase, decarboxylating, EC 1.1.1.40) from T. cruzi has an apparent molecular size of only half that of the mammalian enzyme. The probable subunit structure of these T. cruzi enzymes has been deduced from the molecular weights by comparison with mammalian data. The results are compatible with recent interpretations of isozyme data implying the existence of genetic diploidy in trypanosomes.

Flow cytometry used to distinguish between complete and partial hydatidiform moles.

Cells prepared from fresh tissue from a normal conception, and three complete hydatidiform moles (CHMs), were shown to be diploid by flow cytometry, while triploid populations were identified in tissue from two partial hydatidiform moles (PHMs). Similar results were found in cells from formalin-fixed, paraffin-embedded material. In nine cases where cytogenetic analysis was carried out this correlated with the results from flow cytometry. Using flow cytometry we were able to show that two cases which were pathologically complete were in fact diploid, despite the finding of trisomy at one locus in the molar tissue using biochemical markers. The most likely explanation of apparent trisomy of the enzymes in the molar tissue was contamination by maternal tissue.